MiR-146a-5p deficiency in extracellular vesicles of glioma-associated macrophages promotes epithelial-mesenchymal transition through the NF-κB signaling pathway

Glioma-associated macrophages (GAMs) are pivotal chains in the tumor immune microenvironment (TIME). GAMs mostly display M2-like phenotypes with anti-inflammatory features related to the malignancy and progression of cancers. Extracellular vesicles derived from immunosuppressive GAMs (M2-EVs), the essential components of the TIME, greatly impact the malignant behavior of GBM cells. M1- or M2-EVs were isolated in vitro, and human GBM cell invasion and migration were reinforced under M2-EV treatment. Signatures of the epithelial-mesenchymal transition (EMT) were also enhanced by M2-EVs. Compared with M1-EVs, miR-146a-5p, considered the key factor in TIME regulation, was deficient in M2-EVs according to miRNA-sequencing. When the miR-146a-5p mimic was added, EMT signatures and the invasive and migratory abilities of GBM cells were correspondingly weakened. Public databases predicted the miRNA binding targets and interleukin 1 receptor-associated kinase 1 (IRAK1) and tumor necrosis factor receptor-associated factor 6 (TRAF6) were screened as miR-146a-5p binding genes. Bimolecular fluorescent complementation and coimmunoprecipitation confirmed interactions between TRAF6 and IRAK1. The correlation between TRAF6 and IRAK1 was evaluated with immunofluorescence (IF)-stained clinical glioma samples. The TRAF6-IRAK1 complex is the switch and the brake that modulates IKK complex phosphorylation and NF-κB pathway activation, as well as the EMT behaviors of GBM cells. Furthermore, a homograft nude mouse model was explored and mice transplanted with TRAF6/IRAK1-overexpressing glioma cells had shorter survival times while mice transplanted with glioma cells with miR-146a-5p overexpression or TRAF6/IRAK1 knockdown lived longer. This work indicated that in the TIME of GBM, the deficiency of miR-146a-5p in M2-EVs enhances tumor EMT through disinhibition of the TRAF6-IRAK1 complex and IKK-dependent NF-κB signaling pathway providing a novel therapeutic strategy targeting the TIME of GBM.

INTRODUCTION Glioblastoma (GBM) is a fatal cranial malignant tumor that accounts for almost 57% of all gliomas and exhibits a highly unfavorable outcome [1]. Currently, the recommended GBM therapeutic strategy is surgical resection followed by chemoand radiotherapy [2]. Despite remarkable progress in targeting GBM, the overall prognosis remains poor [3]. Due to its multiple therapeutic resistance, uncontrollable growth, and unpredictable relapse events [4,5]. The tumor immune microenvironment (TIME) comprises various immune cells and numerous soluble inflammatory mediators. It exhibits unique metabolic and immunological features that influence the malignant behaviors and development of GBM [6,7]. Tumor progression is primarily determined by local immune responses linked with cancer cells [8]. However, it has not been easy to uncover the cellular interplay within the glioma TIME due to its complex and multidimensional communication. With the revelation of the connection between tumor-associated macrophages (TAMs) and tumor cells, TAMs have been recognized as one of the most critical components affecting tumor treatment. Nevertheless, due to the high temporospatial heterogeneity and hereditary plasticity, TAM-related immunotherapies have not achieved encouraging efficacy.
Glioma-associated macrophages/microglia (GAMs) account for over half of the GBM tumor bulk's live cells [9]. Via a complex multidimensional regulatory network, GAMs can be polarized toward proinflammatory (M1-like) and anti-inflammatory (M2-like) phenotypes and thus exhibit antitumor or protumor effects, respectively. The M1 and M2 phenotypes are the extreme immune states of activated macrophages representing the common immunological features of infiltrating macrophages [10]. Compared with proinflammatory macrophages, GAMs show both immunostimulatory and suppressive features simultaneously, and promote gliomagenesis, continued tumor growth, and increased malignancy [11][12][13]. Extracellular vesicles (EVs) derived from GAMs are important components of the TIME and contain various cargos, including peptides, lipids, mRNAs, and microRNAs (miRNAs), which are crucial vehicles for communication between tumor cells and GAMs [14,15]. Among these biologically active cargos, miRNAs are small but mighty [16] and greatly impact the progression and recurrence of GBM [17,18].
In this work, based on open online databases, we confirmed that in GBM, immunosuppressive genes related to GAM were notably elevated and that the mRNA profile of GAMs more closely resembled that of the M2 phenotype. We focused on the differences in tumor cell behaviors caused by GAMs with different immunological characteristics and noticed that EVs derived from M2-like GAMs (M2-EVs) induced tumor cell invasion and migration. Comparisons of miRNAs in EVs showed that miR-146a-5p, which damages NF-κB activities and EMT behavior by binding to interleukin 1 receptor-associated kinase 1 (IRAK1) and tumor necrosis factor receptor-associated factor (TRAF6), was deficient in M2-EVs, suggesting that miR-146a-5p could be an encouraging GBM treatment candidate.

RESULTS
Immunosuppressive GAM-associated genes are upregulated in GBM To explore the immune status of the infiltrated macrophages in GBM, we evaluated the differential expression of seven immunostimulatory GAM-related genes (TLR-4, IFNG, IL1B, TNF, NF2, CD80, and CD86) and seven immunosuppressive GAM-related genes (IDO, IL10, TGFB1, CCL2, CCL5, CD163, and MSR1) in patients with LGG and GBM and the relationships of these genes with survival in TCGA database (Fig. 1A, B). All immunosuppressive genes and five immunostimulatory genes were elevated in GBM patients compared with LGG patients. NF2 and TLR4 were downregulated in GBM. Overall survival (OS) and disease-free survival (DFS) were not influenced by the expression of immunostimulatory genes in these two groups (Fig. 1C). However, when the seven immunosuppressive genes were highly expressed, the OS and DFS times were significantly decreased. (Fig. 1D). To verify the findings in clinical cases, CD163 and MSR1 expression was evaluated by immunohistochemical (IHC) in 12 human glioma specimens. The numbers of CD163-and MSR1positive cells were higher in the GBM slices than in grade II and grade III glioma specimens (Fig. 1E). These findings indicated that infiltrating GAMs exhibit more immunosuppressive features in high-grade gliomas and that high expression of immunosuppressive genes is tightly related to GBM progression and unfavorable outcomes.
Coculture with M2-EVs enhanced U87MG and A172 cell invasion and migration EVs are the most critical vesicles for communication between GAMs and GBM cells and are indispensable components of the TIME. We found that immunosuppressive genes were upregulated in GBM, and M2-like GAMs were established to simulate the immunosuppressive characteristics of GAMs, while M1-like GAMs were established as a control. To verify that EVs derived from immunosuppressed GAMs are the main contributor to GBM malignant behaviors, THP-1 cells simulated immunostimulatory (M1-like) and immunosuppressive (M2-like) GAM models in vitro under GM-CSF or IL-4/IL-13 stimulation. Real-time polymerase chain reaction (RT-PCR) was conducted to evaluate the mRNA levels of TNF-α, TLR-4, CCL-2, and TGF-β to validate the immunological features of M1-and M2-like GAMs. TNF-α and TLR-4 were upregulated in M1-like GAMs and downregulated in M2-like GAMs. In contrast, CCL-2 and TGF-β were downregulated in M1-like GAMs and upregulated in M2-like GAMs ( Fig. 2A), suggesting that M1-like GAMs were in an immunostimulatory state and M2-like GAMs were in an immunosuppressive state. EVs derived from M1-or M2-like GAMs (M1-or M2-EVs, respectively) were isolated and found to have a 30-150 nm-diameter bilayer membrane biconvex disc-shaped structure by TEM (Fig. 2B) and nanoparticle tracking analysis (NTA; Fig. 2C). The specific markers CD63, CD9, and CD81 were detected in EVs and conditioned medium by western blotting (Fig. 2D), which ensured the purity of the EVs. After labeling with Dil, M1-EVs and M2-EVs were cocultured with U87MG cells whose cytoskeleton was stained with phalloidin, and red fluorescence was observed in the cytoplasm (Fig. 2E). Collectively, these findings demonstrated that M1-EVs and M2-EVs were successfully extracted from the medium supernatant and were able to be internalized into GBM cells.
Subsequently, the EMT behaviors of GBM cells were assessed under coculture with M1-and M2-EVs. Transwell (Fig. 2F) and wound healing assays (Fig. 2G) were conducted, and both U87MG and A172 cells cocultured with M2-EVs were more invasive than those cocultured with M1-EVs and displayed rapid wound closure within 72 h. Markers of EMT, including N-cadherin, MMP-2, and vimentin, were evaluated by Western blotting (Fig. 2H). Ncadherin, MMP-2, and vimentin were expressed at high levels in M2-EV-treated U87MG and A172 cells compared with cells treated with M1-EVs. In combination, our work suggested that compared with M1-EVs, M2-EVs increased GBM cell migratory and invasive abilities promoting EMT in these cells in vitro.

MiR-146a-5p is deficient in M2-EVs
In EV-dependent biological regulation, miRNAs are the prominent cargo and exert substantial impacts [19]. To determine the specific compositions and underlying mechanisms that lead to the functional differences between the effects of M1-EVs and M2-EVs on GBM cells, MiRNA sequencing (miRNA-Seq) was performed. The differential expression of miRNAs derived from M1-and M2-EVs was analyzed with DEGseq. There were 237 miRNAs with the most marked differences (Fig. 3A and  Supplementary Table S1), and miR-146a-5p was selected (Fig. 3B) as a candidate. MiR-146a-5p levels among M0, M1-, and M2-like GAMs were estimated with RT-PCR and were higher in M1-like GAMs than in M2-like GAMs (Fig. 3C), confirming that miR-146a-5p was lower in M2-like GAMs.
Exosomal miR-146a-5p promoted EMT in GBM cells To verify whether malignant EMT behaviors are influenced by miR-146a-5p derived from GAM-EVs, migratory and invasive behaviors and the expression of EMT markers were evaluated in GBM cells. The miR-146a-5p mimic and inhibitor and their negative control (NC) were transfected into U87MG and A172 cells. The miR-146a-5p mimic reduced their migration and invasion in Transwell assays of U87MG and A172 cells, and decreased the wound healing ability, and cells with miR-146a-5p inhibitors were more capable of migrating and invasive ( Fig. 3D-G). Furthermore, the levels of N-cadherin, MMP-2, and vimentin were significantly elevated when the miR-146a-5p inhibitor was applied to the cells. When these cells were treated with the miR-146a-5p mimic, the levels of N-cadherin and MMP-2 were markedly decreased (Fig. 3H).
MiR-146a-5p binds to IRAK1 and TRAF6 To clarify the potential downstream mechanisms of miR-146a-5p, six public online databases were used to predict its binding genes. Nine genes were then considered potential candidates. The relationships of these nine genes and their encoded proteins were analyzed with STRING, and it was found that TRAF6 and IRAK1 had stronger connections (Fig. 4A). According to the numbers of binding sites for miR-146a-5p predicted with TargetScan, TRAF6 has three conserved sites, and IRAK1 has two conserved sites. Therefore, IRAK1 and TRAF6 were selected as promising targets. Then, dual-luciferase reporter assays were executed to test whether miR-146a-5p was directly bound to these genes. TRAF6 and IRAK1 WT and MT 3′UTR-driven Fig. 1 Immunosuppressive glioma-associated macrophage (GAMs)-related genes were upregulated in glioblastoma multiforme (GBM) compared with low-grade glioma (LGG). A The mRNA expression of the immunostimulatory genes CD80, CD86, IFNG, IL1B, NF2, TNF, and TLR4 in LGG and GBM in The Cancer Genome Atlas (TCGA) database was analyzed. B The mRNA expression of the immunosuppressive genes CCL2, CCL5, CD163, IDO1, IL10, MSR1, and TGFB in LGG and GBM in TCGA was analyzed. C The overall survival (OS) and disease-free survival (DFS) of GBM and LGG patients in TCGA with differential expression of CD80, CD86, IFNG, IL1B, NF2, TNF, and TLR4 was analyzed with GEPIA 2.0. D The OS and DFS of patients with GBM and LGG in TCGA with differential expression of CCL2, CCL5, CD163, IDO1, IL10, MSR1, and TGFB was analyzed with GEPIA 2.0. E The levels of TLR4, CD86, CD163, and MSR1 in GBM (n = 4) and LGG (grade II and III gliomas, n = 8) clinical samples were determined by immunohistochemical (IHC) staining and quantitative analysis. Scale bar = 50 μm. *p < 0.05, **p < 0.01, ***p < 0.001. pmirGLO luciferase vectors were constructed (Fig. 4B), and the miR-146a-5p mimic was cotransfected into U87MG cells along with these vectors. The miR-146a-5p mimic inhibited the relative luciferase activity of these WT 3′UTRs compared to the control. In contrast, the luciferase activity of these MT 3′UTRs was not influenced by the miR-146a-5p mimic (Fig. 4C). Furthermore, M1-EVs were added to U87MG cells transfected with TRAF6 and IRAK1 WT and MT 3′UTR-driven pmirGLO luciferase vectors alone. The results showed that M1-EVs inhibited the luciferase activities of WT 3′UTR-driven luciferase (Fig. 4D). To detect the C. Xu et al.
impact of M1-EVs and M2-EVs on the expression of TRAF6 and IRAK1, these EVs were added to U87MG and A172 cells. The expression level was detected by western blotting and showed that M2-EVs upregulated TRAF6 and IRAK1 compared with M1-EVs (Fig. 4E). Then, the miR-146a-5p mimic or inhibitor was transfected into U87MG and A172 cells and the mimic reduced the levels of TRAF6 and IRAK1, while the inhibitor increased their levels (Fig. 4F).
Furthermore, to clarify the expression of these two genes in glioma, we retrieved the data in TCGA and found that compared with LGG, the expression of IRAK1 and TRAF6 was upregulated in GBM ( Supplementary Fig. S1). Both LGG and GBM express TRAF6 positively correlated with IRAK1 ( Fig. 4G and Supplementary Fig.  S2A) at the transcriptional level. We used TRAF6 and IRAK1 as a gene set and evaluated its effect on the survival time of glioma patients in TCGA. The LGG patients received longer survival times when they expressed lower levels of TRAF6 and IRAK1 but GBM patients did not obtain a remarkable benefit ( Fig. 4H and Supplementary Fig. S2B), which might be caused by a large number of infiltrating M2-like GAMs rather than M1-like GAMs. These results confirmed that TRAF6 and IRAK1 are the targets of miR-146a-5p. Low IRAK1 levels increased the OS of LGG and overall glioma patients ( Supplementary Fig. S2C) indicating that IRAK1 might be the direct effector modulating the progression of glioma.
Since the miRNA data of GBM patients were not available in TCGA database, we analyzed the correlation of miR-146a-5p vs. TRAF6 and vs. IRAK1 in the Chinese Glioma Genome Atlas (CGGA) database. However, the expression of miR-146a-5p was not negatively correlated with TRAF6 and IRAK1 in either LGG or GBM patients ( Supplementary Fig. S3A), which seemed inconsistent with the changes we found in vitro. In addition, the OS of patients with different miR-146a-5p levels did not show a significant difference despite the longer survival time tendency presented in the high miR-146a-5p group ( Supplementary  Fig. S3B). We deduced that the intratumoral heterogeneity of glioma might lead to such inconsistencies of bulk RNA sequencing.
TRAF6-IRAK1 complex was the regulatory target of miR-146a-5p It has been reported that complicated posttranslational modifications (PTM) exist between TRAF6 and IRAK1. The correlation between these two genes at the protein level was evaluated. Cell lines with TRAF6 and IRAK1 being knockdown by shRNA were established, and cell lines with overexpression of these two genes and miR-146a-5p were also established at the same time.
Overexpression of miR-146a-5p decreased the protein levels of TRAF6 and IRAK1 (Fig. 5A). Obviously, when TRAF6 was overexpressed, IRAK1 was significantly degraded and when IRAK1 was overexpressed, TRAF6 was inhibited. However, when TRAF6 was knocked down, we detected lightly decreased IRAK1, and when IRAK1 was knocked down, the expression of TRAF6 was remarkably increased (Fig. 5B). There was no significant change at the mRNA level ( Supplementary Fig. 4A). GBM data from the Clinical Proteomic Tumor Analysis Consortium (CPTAC) were analyzed, and we found that the protein expression levels of these two molecules were highly expressed in GBM and also positively correlated despite insignificant differences ( Fig. 5C and Supplementary Fig. 4B). The protein levels of TRAF6 and IRAK1 were estimated with IF-stained glioma samples. Two GBM samples were detected with double-labeled IF and the distribution of positive cells presented specific spatial features (Fig. 5D). TRAF6 was expressed at high levels in the cellular tumor zone and pseudopalisading cells around necrosis while in the microvascular proliferation zone and cellular tumor zone, IRAK1 was expressed at high levels. We referred to RNA-seq data in the Ivy Glioblastoma Atlas Project (Ivy GAP) and found that there were differences between the expression of these two genes in different areas ( Fig. 5E and Supplementary Fig. 4C). Then, the 8 LGG samples and 4 GBM samples were stained with TRAF6 or IRAK1 at the continuously adjacent position, and the positive scores (defined by the fluorescence intensities and ratio of positive cells) of TRAF6 and IRAK1 in the GBM group were higher than those in the LGG group. Meanwhile, the positive scores of TRAF6 and IRAK1 were positively correlated (Fig. 5F, G and Supplementary Fig. S4D).
miR-146a-5p and TRAF6-IRAK1 Expressions influenced the progression of GBM To validate the influence of TRAF6 and IRAK1 on GBM progression in vivo, an allogeneic U87MG-Luc GBM mouse model was employed. To simulate the impact of miR-146a-5p, U87MG-Luc NC , U87MG-Luc miR-146a-5p , U87MG-Luc sh-NC , U87MG-Luc ov-NC , U87MG-Luc sh-TRAF6/IRAK1 , and U87MG-Luc ov-TRAF6/IRAK1 cells were implanted into nude mice in situ. Three weeks after implantation, tumors were detected in vivo with a small animal live imaging system. Compared with control GBM tumors, the intensities of mice with U87MG-Luc miR-146a-5p GBM cells were significantly weaker than those of mice with U87MG-Luc NC . U87MG-Luc sh-TRAF6/IRAK1 GBM tumors were smaller with weaker luminescence intensities, and U87MG-Luc ov-TRAF6/IRAK1 tumors were larger and had higher luminescence intensities (Fig. 7A, B). H&E staining showed that U87MG-Luc miR-146a-5p and U87MG-Luc sh-TRAF6/IRAK1 tumors had clear boundaries and surrounding normal tissue was suppressed, while U87MG-Luc ov-TRAF6/IRAK1 tumors infiltrated into normal tissue with a blurred boundary (Fig. 7C, D). Moreover, the survival of mice bearing these different GBM tumors differed significantly (Fig. 7E, F). All mice bearing U87MG-Luc miR-146a-5p tumors were alive after~6 weeks. No mice bearing U87MG-Luc ov-TRAF6/IRAK1 GBM tumors were alive after approximately 4 weeks. More than half of the mice bearing U87MG-Luc sh-TRAF6/IRAK1 GBM tumors were alive while only one mouse bearing U87MG-Luc sh-NC GBM tumors was alive at the end of the experiment (7 weeks). The HR of ov-TRAF6/ IRAK1 was 13.03, illustrating that the deficiency of miR-146a-5p and high expression of TRAF6-IRAK1 are important risk factors for GBM progression.

DISCUSSION
This work demonstrated that M2-EVs promoted GBM progression due to miR-146a-5p deficiency, which was elevated in M1-EVs. MiR-146a-5p deficiency in GAM-EVs reduced the invasion and migration of GBM cells through disinhibition of the TRAF6-IRAK1 complex and its downstream IKK-γ dependent NF-κB signaling pathway. In this study, we obtained a better understanding of GAMs with different immunological characteristics on the malignant progression of GBM.
In the past, the effect of GAMs on GBM has been widely studied. However, the complicated milieu and its interactions remain poorly understood. Single-cell RNA (scRNA) sequencing revealed that macrophages are the most common immune cell type in GBM [6,22]. Due to the significant heterogeneity of GAMs, which are educated by the TIME [23,24], it is difficult to precisely determine the activation states and transcriptional signatures of GAMs. Analysis of clinical specimens showed that GBMs contained a mixture of M1/M2-like GAMs [25]. We examined the mRNA levels of typical immunostimulatory and immunosuppressive gene markers from public databases and clinical samples and showed that immunosuppressive macrophage-related genes were upregulated in GBM compared with LGG and that the immunosuppressive gene sets significantly influenced the survival time of GBM patients, which was consistent with previous studies [26]. Gene coexpression network analysis based on single-cell profiles confirmed that GAMs, especially those with the M2-like phenotype, are negatively associated with glioma survival [27]. Accordingly, it is necessary to evaluate the function of GAMs during the progression of GBM based on their immunological features. The immunosuppressive characteristics of GAMs are the predominant results of education by the TIME of GBM [28]. The establishment of M1-and M2-like GAMs in vitro helped analyze the differences in GAMs and explore key targets under these opposite immune environments during our study.
EVs derived from GAMs are one of the most important vehicles for communication between GAMs and GBM cells. Recently, the effect of GBM-related EVs and their cargos has attracted increasing attention in terms of diagnosis [29], the exploration of underlying mechanisms [30], and the exploitation of new therapies [31]. However, studies focused on GAM-EVs are still in the initial stage, although the role of tumor modulators of GAMs is widely accepted [26]. Since it is challenging to isolate EVs derived from specific GAMs from clinical samples, studies lack data helpful for mapping the overall landscape of GAM-EVs in vivo, which hinders progression in the exploration of GAM-EVs. Furthermore, the mechanism by which cargos are loaded into EVs remains unclear. Specifically, increasing the expression of specific miRNAs and proteins in parent cells may not increase their contents in EVs, which limits the modulation of EVs in vivo, although the development of engineered exosomes has progressed dramatically [32]. By seeking to identify the main cargos in EVs, we hope that these cargos can be targeted directly in future studies.
Various miRNAs in EVs are effectors that regulate the biological behaviors of GBM. Research has shown that a series of miRNAs enhanced the transition of glioma stem cells from proneural to mesenchymal [33]; that miR-21 facilitates the invasion, proliferation, and migration of glioma cells [34]; and that miR-92a and miR-15a in M2-EVs impaired the abilities of invasion and migration in GBM cells [35]. In our study, to identify valuable miRNAs, we compared the differences in the types and quantities of miRNAs derived from M1-EVs and M2-EVs. MiR-146a-5p is considered an Fig. 3 Compared to M1-EVs, miR-146a-5p was deficient in M2-EVs and enhanced U87MG and A172 cell invasion and migration behaviors. A The heatmap showed the differential expression of miRNAs between M1-and M2-EVs according to miRNA-sequencing. B The volcano graph plot showed the relationship between the fold change and the significance of differentially expressed miRNAs of M2-EVs compared to M1-EVs. C The miR-146a-5p level of M0, M1-like GAMs, and M2-like GAMs was evaluated by RT-PCR. MiR-146a-5p was significantly increased in M1-like GAMs compared with M0 and M2-like GAMs. D, E Transwell assays and quantitative analysis were performed to evaluate the migratory and invasive abilities of U87MG cells and A172 cells transfected with the miR-146a-5p mimic, miR-146a-5p inhibitor or the corresponding negative control sequence (NC) for 24 h. F, G Wound healing assays and quantitative analysis were used to evaluate the migration ability of U87MG cells and A172 cells transfected with the miR-146a-5p mimic, miR-146a-5p inhibitor, mimic NC, or inhibitor NC for 24 h. H Western blotting analysis of the expression of E-cadherin, N-cadherin, MMP-2, and vimentin in U87MG and A172 cells transfected with the miR-146a-5p mimic, miR-146a-5p inhibitor, mimic NC, or inhibitor NC. *p < 0.05, **p < 0.01, ***p < 0.001.
adaptive immunity-related miRNA. The human MIR146A gene, which is highly upregulated when exposed to IL-1β, LPS, and TNFα, is observed to be NF-κB responsive in cells [36]. Healthy aging and inflammation are improved by miR-146a via its influence on canonical and noncanonical NF-κB pathways [37]. It critically impacts oncogenesis and tumor progression in various cancers [38]. MiR-146a-5p is regarded as an antitumor molecule in numerous cancers, such as esophageal cancer and CML, but is considered an oncomiR in bladder and cervical cancers, as well as melanoma. Previous research has confirmed that overexpression of miR-146a-5p in glioma cells led to inhibited growth [39] and enhanced apoptosis [40]. These results showed that the progression of GBM can be limited by miR-146a-5p, similar to our results. Furthermore, we revealed that immunostimulatory GAMs were a vital source for miR-146a-5p and that its level was notably lower in M2-EVs than in M1-EVs. MiR-146a-5p deficiency in both GBM cells and TIME is one of the deterministic factors that promotes GBM progression.
In this work, miR-146a-5p derived from GAM-EVs inhibited the EMT behaviors of GBM cells by inhibiting the activation of the TRAF6-IRAK1 complex. IRAK1 and TRAF6 are classical miR-146a target genes, although their relationship seemed to be conditional [41]. As we found in this work, miR-146a-5p influenced the expression of TRAF6 and IRAK1 at both mRNA and protein levels in vitro, although the analyzation on silicon is inconsistency. But the interaction between TRAF6 and IRAK1 was complicated. In both LGG and GBM, according to the analysis of public transcriptome databases, the TRAF6 and IRAK1 expression levels were positively correlated. And in the GBM protein database, TRAF6 and IRAK1 were not remarkably correlated. Interestingly, we established TRAF6 or IRAK1 knockdown and overexpression cell lines and evaluated the mRNA expression of these genes and found that the changes in TRAF6 and IRAK1 expression were not noticeably reciprocally influenced, while the changes were significant at the protein level. TRAF6 is an E3-ubiquitin ligase, and IRAK1 is regarded as the substrate for TRAF6 [21,42]. TRAF6 is able to catalyze the formation of polyubiquitin linked at both Lys63 and Lys48 [42] in IRAK1. Lys48-linked ubiquitination facilitates subsequent proteasomal degradation in the ubiquitinproteasome pathway while TRAF6-induced Lys63 ubiquitination is necessary for IRAK1-induced activation of the NF-κB signaling pathway [43]. Whereas, during this interaction, TRAF6 is also conditional downregulated through a proteasome-dependent pathway that is facilitated by IRAK1, which depends on the distance between the N-terminal death domain and the TRAF6 -binding site in IRAK1 [44,45]. Meanwhile, IRAK1 promotes oligomerization of TRAF6. It is regarded as a brake of excessive activation of the NF-κB pathway. During our observation, the expression pattern of TRAF6 and IRAK1 in GBM samples were not banded together. Both of them have specific spatial expression characteristics, which explains why there was no significant correlation between TRAF6 and IRAK1 at the protein level in GBM tissue. Compared with the leading edge of GBM, cellular tumors expressed more TRAF6 and IRAK1 enhanced the activation of the NF-κB signaling pathway and then increased the invasive ability of GBM cells.
At the transcriptome level, bulk mRNA sequencing data hardly reflect the features led by high heterogeneity, although we noticed that the mRNAs of TRAF6 and IRAK1 were positively correlated. Only one GBM case in Ivy GAP had high miR-146a levels, and the expression levels of TRAF6 and IRAK1 were relatively lower. We observed that miR-146a-5p binding diminished the expression of both IRAK1 and TRAF6 in vitro. All of the above results illustrate that miR-146a-5p is capable of regulating the TRAF6-IRAK1 complex and its downstream NF-κB signaling pathway.
We suggest that miR-146a-5p is in a one-to-multi state with its binding genes, although previous studies hypothesized that the relationships between miRNAs and their target genes are oneto-one or multi-to-one. The deterministic factor of this modification is the quantity of miRNA according to our conjecture, which needs further validation. When TRAF6 and IRAK1 were knocked down simultaneously, NF-κB signaling pathway activation and EMT marker expression were significantly inhibited. Compared with single-gene knockdown, dual-gene silencing more reliably simulated EVs miR-146a-5p biological function, and marked phosphorylation of the IKK-γ and NF-κB p65 subunits was observed.
Recently, M1-EVs have been considered carriers in GBM therapies, although a specific cargo was not mentioned in this study [46]. Repolarizing M2-like GAMs into M1-like GAMs with effective biomaterials (such as a virus-mimicking membranecoated nucleic acid nanogel Vir-Gel [47] and bovine serum albumin-MnO2-SAL [48]) or active molecules (such as Galectin-9 siRNA [49] and curcumin [50]) that were able to be transferred into the TIME and to exert therapeutic functions were considered a promising approach for GBM treatment. Our findings demonstrated that elevating miR-146a-5p levels in the TIME might be a novel therapeutic approach for GBM.

Patient samples
Samples were collected from twelve patients who had surgically resected gliomas between 2013 and 2023 at Chongqing General Hospital and were histologically confirmed postoperatively as glioma. The assigned glioma grades were based on pathological diagnosis under the WHO Classification of Tumors, 5th Edition, Volume 6: Tumors [51]. The Ethics Committee of Chongqing General Hospital supervised and approved this study in compliance with the Declaration of Helsinki. Patients (or their representatives) who were involved signed the individual informed consent form.

Cell lines and reagents
U87MG and A172 cells, the human GBM cell lines, were cultured with DMEM (Gibco, Carlsbad, USA) with 10% fetal bovine serum (FBS; Gibco), penicillin 50 units/ml, and streptomycin 50 μg/ml (Beyotime Fig. 4 TRAF6 and IRAK1 are the miR-146a-5p target genes in GBM. A Venn diagram (left panel) showed the intersection of predicted binding genes of miR-146a-5p obtained from 6 online public databases. Nine genes (middle panel) were predicted in all six databases. Association analysis of the proteins encoded by these 9 genes performed with STRING (right panel) showed a strong connection between TRAF6 and IRAK1. B Schematic representation of the TRAF6 3′UTR and IRAK1 3′UTR. MT sequences were generated at the 3′UTR binding sites of miR-146a-5p according to the prediction. C Relative luciferase activity was determined in U87MG cells that were cotransfected with the miR-146a-5p mimic or mimic NC and the WT-3′UTR or MT-3′UTR of TRAF6 or IRAK1. D Relative luciferase activity was determined in U87MG cells transfected with the WT-3′UTR or MT-3′UTR of TRAF6 or IRAK1 for 24 h and then cultured with M1-EVs or mock. E Western blotting measured the expression levels of TRAF6 and IRAK1 in U87MG and A172 cells cocultured with M1-EVs or M2-EVs. F Expression of TRAF6 and IRAK1 in U87MG and A172 cells transfected with the miR-146a-5p mimic, miR-146a-5p inhibitor, mimic NC, or inhibitor NC. G The correlation of TRAF6 and IRAK1 mRNA expression in patients with LGG and GBM in TCGA was estimated, and the mRNA expression level of TRAF6 was positively correlated with that of IRAK1 in GBM and LGG. H The DFS and OS of glioma patients with different TRAF6/IRAK1 expressions were analyzed in TCGA database. Both DFS and OS of LGG patients with higher TRAF6/IRAK1 expression levels were shorter than those with lower expression levels. Both DFS and OS of GBM patients did not obtain significant benefit.

EV extraction
EVs were extracted from the FBS-free conditioned medium of M1-GAMs and M2-GAMs and extracted by ultracentrifugation with an Optima MAX-XP instrument (Beckman, Indianapolis, USA) as previously described [53,54]. In brief, the cell culture supernatant was first centrifuged at 300×g for 10 min to remove whole cells, and then a 2000×g centrifuge for 10 min was performed to remove dead cells. To remove cell debris, the supernatant was ultracentrifuged for 30 min at 10,000×g and ultracentrifuged for 70 min at 100,00×g to isolate EVs. Finally, the pellet was washed with phosphate-buffered saline (PBS). Retained EVs were then analyzed and used for follow-up studies.

Transmission electron microscopy and nanoparticle tracking analysis
Transmission electron microscopy (TEM) was used to observe the morphological features of EVs that were negatively stained. After being fixed with 1% glutaraldehyde and washed, EVs were placed on Formvar/ carbon-coated 300-mesh copper grids and incubated at room temperature for 1 min. Uranyl acetate stained EVs for 1 min. Images were acquired by TEM (JEM-1400Plus, JEOL, Tokyo, Japan) after the grids were washed and dried.
The concentration of EVs and their size distribution were estimated by NTA (NanoSight NS300, Malvern Panalytical, Malvern, UK). The detailed procedure followed the manufacturer's instructions, as previously described [55]. Data were analyzed with Zetasizer software (Malvern Instruments).

EV internalization
To confirm that M1-EVs and M2-EVs were internalized by GBM cells, EVs were stained and labeled with Dil (Beyotime Biotechnology) following the manual and visualized with an LSM880 laser confocal microscope (Zeiss, Cambridge, UK). They were ultracentrifuged at 100,000×g for 60 min to remove residual dye and resuspended. Then, U87MG cells were cocultured with Dil-labeled EVs for 12 h. After refreshing the culture medium, Alexa Fluor™ 488 phalloidin (Invitrogen, Waltham, USA) and Hoechst (Beyotime Biotechnology) were added to label cells and EV internalization was observed after washing.

MiRNA sequencing of EVs
Isolated M1-EVs and M2-EVs were subjected to miRNA-Seq analysis (GCBI, Shanghai, China). The differential expression of miRNAs was analyzed with DEGseq and uploaded to the NCBI Gene Expression Omnibus (GEO) database (GSE212171, www.ncbi.nlm.nih.gov/geo). Heatmaps showing the differentially regulated miRNAs were generated using online tools provided by GCBI (https://biosys.bgi.com/), and a volcanic plot was developed to visualize the significance and change fold of EV miRNAs.

Dual-luciferase reporter assay
Two fragments of the wild-type (WT) TRAF6 3′ untranslated region (UTR), one fragment of the WT IRAK1 3′UTR, and the corresponding MT sequences were used to construct the WT and mutant (MT) pmir-GLO luciferase vectors. The plasmids containing the WT TRAF6 or IRAK1 3′UTR or the corresponding 3′ UTRs with miRNA binding site mutations driven by the pmirGLO luciferase reporter and the mimic NC and has-miR-146a-5p mimic were cotransfected in U87MG cells with Lipo 2000. After 24 hours, the relative luciferase activity was detected by a Dual-Luciferase Reporter Assay Kit (Promega, Madison, USA) and a Varioskan Flash microplate reader (Thermo Scientific, Waltham, USA) following the manufacturer's instructions.
TRAF6 and IRAK1 knockdown and overexpression, and has-miR-146a-5p overexpression ShRNA plasmids were used to interfere with the translation of TRAF6 and IRAK1. The TRAF6 and IRAK1 shRNA sequences were inserted into pLVX-shRNA2 (Clontech 632179, Mountain View, USA). The vector pLVX-CMV-IRES-PURO was employed to construct the TRAF6 and IRAK1 overexpression plasmids. (The primer and shRNA sequences are listed in Supplementary Table S4). The vector GV298 (U6-MCS-Ubiquitin-Cherry-IRES-puromycin) was employed to construct the has-miR-146a-5p overexpression plasmid. All plasmids were transformed into E. coli strain Stbl3. The lentivirus was then packaged in HEK293T cells with psPAX2 (Addgene #12260) and pMD2.G (Addgene #12259). After transfection, HEK293T cells were incubated for 48 h. The supernatant with virus particles was collected. After being filtered, the virus particles were added to infect U87MG and A172 cells for infection. Successfully infected cells were screened with puromycin. To validate the knockdown or overexpression of IRAK1 and TRAF6, Western blot assays were conducted.  Complicated interactions exist between TRAF6 and IRAK1. A, B Western blotting was used to detected the expression of TRAF6 and IRAK1 at protein levels in U87MG cells with miR-146a-5p overexpression, and TRAF6 or IRAK1 knockdown, and TRAF6 or IRAK1 overexpression. C The expression correlations of TRAF6 and IRAK1 at protein levels in GBM (n = 99) and normal brain tissue (n = 10) were analyzed in CPTAC databases. D Slides derived from two GBM samples were evaluated with TRAF6 (green) and IRAK1 (red) dual-labeled immunofluorescence (IF). H&E staining was used to evaluate the pathological features and to identify specific areas (adjacent area not the same zone). Left panel showed the microvascular proliferation area and right panel showed the leading edge (scale bar = 20 μm). The scale bar of the merged panoramic image is 2000 μm. E The expressions of TRAF6, IRAK1, and miR-146a at mRNA levels in different GBM areas were analyzed with Ivy GAP database. F The levels of TRAF6 and IRAK1 in GBM (n = 4) and LGG (grade II and III gliomas, n = 8) clinical samples were determined by IF staining and positive scores were of each sample based on the fluorescence intensities and numbers of positive cell. The positive scores of TRAF6 and IRAK1 in GBM were remarkably higher than those in LGG. G The Person correlation was used to analyze the correlation of TRAF6 and IRAK1 expressions in clinical samples at protein levels with the positive scores. H BiFC was used to estimate the spatial formation of the TRAF6-IRAK1 complex. When pBiFC-VN173-TRAF6 and pBiFC-CC155-IRAK1 or pBiFC-VN173-IRAK1 and pBiFC-CC155-TRAF6 were cotransfected into U87MG cells, green fluorescence was detected in the cytoplasm. Scale bar = 50 µm. I Coimmunoprecipitation (CoIP) was performed to evaluate interactions between TRAF6 and IRAK1 at the protein level. The anti-IRAK1 antibody immunoblotted (IB) the TRAF6 that immunoprecipitated (IP) from total protein, while the IRAK1 IP from cell lysates and IB with an anti-TRAF6 antibody. Fig. 6 The miR-146a-5p regulated TRAF6-IRAK1 complex and the IKK-dependent NF-κB pathway. Wound healing (A, C) and transwell assays (B, D) were used to estimate the invasive and migratory abilities of U87MG miR-146a-5p , U87MG ov-TRAF6/IRAK1 , and U87MG sh-TRAF6/IRAK1 cells compared with the corresponding controls. Scale bar = 200 μm. E, F Western blotting analysis as used to evaluate the levels of E-cadherin, Ncadherin, MMP-2, and vimentin in U87MG miR-146a-5p , U87MG ov-TRAF6/IRAK1 , and U87MG sh-TRAF6/IRAK1 cells and their corresponding controls. G, H Western blotting was conducted to measure the expression of IKKγ, phosphorylated-IKKγ (p-IKKγ), IKBα, p-IKBα, p65, and p-p65 in U87MG miR-146a-5p , U87MG ov-TRAF6/IRAK1 , and U87MG sh-TRAF6/IRAK1 cells and their corresponding controls. The ratios of p-IKKγ/IKKγ, p-IKBα/IKBα, and p-p65/p65 were calculated. n = 3, *p < 0.05, **p < 0.01, ***p < 0.001. and extension at 72°C for 30 s. The primer sequences are listed in Supplementary Table S3.

Western blot assays
Prechilled RIPA buffer (Beyotime Biotechnology) was used to prepare total protein. Extracted protein was separated by SDS-PAGE, and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% nonfat milk for 2 h at room temperature and incubated with specific primary antibodies (information regarding the primary antibodies is listed in Supplementary Table S5) at 4°C overnight. Then, corresponding HRPconjugated secondary antibodies (Beyotime Biotechnology) were used in the membrane incubation at room temperature for 1 h. Enhanced chemiluminescence was used for visualization. The original data are available in the Supplementary Material.

Wound healing assay and Transwell assay
Wound healing assays were applied to estimate GBM cell migratory ability. U87MG and A172 cells were seeded and cultured in six-well plates until 100% confluence. These cells were cultured with FBS-free medium for 24 h before the experiment. The wounds were scratched with 200 μL pipette tips, and then the cells were washed 3 times and cultured with FBS-free medium. Wound healing was observed at 72 h with an optical microscope (IX71, Olympus, Tokyo, Japan). ImageJ software was used to estimate and quantify the healing rates.
Transwell assays were used to estimate GBM cell migratory and invasive abilities with chambers that have 0.4 μm micropores (Corning Life Sciences, NY, USA). For invasion assays, U87MG and A172 cells were seeded in the upper chambers of each insert, which contained a Matrigelcoated membrane (Corning Life Sciences), and cultured with FBS-free medium. Medium containing 10% FBS was added to the lower chambers of the 24-well plates. For migration assays, GBM cells were seeded in the upper chambers without Matrigel coating on the membrane. After incubation for 15 h at 37°C and fixation in paraformaldehyde, these cells were stained with crystal violet solution. Afterward, the number of cells on the lower surface of each membrane was determined and averaged in 5 random fields.

Coimmunoprecipitation
Total protein was prepared with Co-IP lysis buffer (Beyotime Biotechnology). Equal amounts of total protein from each sample were incubated with primary antibodies (information regarding the primary antibodies is listed in Supplementary Table S5) at 4°C overnight. Then, Protein A/G PLUS-Agarose (Santa Cruz Biotechnology, Dallas, USA) was added and incubated with shaking at 4°C for 3 h. The bound proteins were eluted with SDS-loading buffer after washing. Immunoprecipitated proteins were used in immunoblotting following procedures described in the Western blotting section above. (Information regarding the primary antibodies is listed in Supplementary Table S5).

Orthotopic homograft mouse model
The animal experimental procedures were supervised and approved by the ethics committee of Chongqing General Hospital. Male BALB/c-nude mice (4-5 weeks) were used in the research. Forty-four mice were randomly divided into four groups, and 5 μL of U87MG-Luc NC , U87MG-Luc ov-miR-146a-5p , U87MG-Luc sh-NC , U87MG-Luc ov-NC U87MG-Luc ov-TRAF6/IRAK1 , or U87MG-Luc sh-TRAF6/IRAK1 cells (1 × 10 5 ) was injected in situ into the brain by using a small animal stereotactic injector (RWD, Shenzhen, China). Mice were fed for 3 weeks, and the tumor sizes and luminescence intensity were measured with a small animal live imaging system (IVIS® Spectrum CT, PerkinElmer, USA). The survival times of mice bearing GBM tumors were recorded, and the tumors were harvested for detection of tumor invasion and analysis of protein expression.
Immunohistochemical, immunofluorescence, and H&E staining GBM specimens obtained from patients were used to detect CD163, CD206, TRAF6, and IRAK1 using IHC staining. The primary steps were as follows: fixation with paraformaldehyde, embedding in paraffin, dewaxing in xylene, rinsing in graded ethanol solutions and rehydration. Next, antigen retrieval was performed and the slides were pretreated with sodium citrate buffer at 95°C for 15 min. After washing with PBS 3 times, the slides were stained with primary antibodies against CD163 and MSR1 (information regarding the primary antibodies is listed in Supplementary  Table S5) at 4°C overnight. After being washed again, the slides were immersed in buffer containing HRP-conjugated anti-mouse/rabbit antibodies for 2 h. DAB chromogen solution was added and then incubated for approximately 1 min. The numbers of CD163-and MSR1-positive cells in 5 random fields were determined and analyzed in images on the same magnification scale.
Frozen GBM slides obtained from patients were used to detect TRAF6 and IRAK1 using IF staining. The slides were fixed with paraformaldehyde, and permeated with Tween-20. After washing with PBS 3 times, the slides were blocked with goat serum and stained with primary antibodies against TRAF6 and IRAK1 (information regarding the primary antibodies is listed in Supplementary Table S5) at 4°C overnight. After being washed again, the slides were stained with CoralLite488-conjugated anti-mouse antibody or CoralLite594-conjugated anti-rabbit antibody for 2 h at room temperature. DAPI was used to stained nucleus for 10 min.
GBM slides derived from patients and brain samples harvested from mice bearing GBM tumors were processed into paraffin sections and stained with H&E to observe the morphological features. For H&E staining, deparaffinization was completed with dimethylbenzene, followed by 3 min of hematoxylin and eosin staining. Then, the stained slices were dehydrated and visualized.

Statistical analysis
All in vitro experiments were performed 3 times independently. IBM SPSS software 24.0 was used for the statistical analysis. Means and standard deviations were used to represent data (means ± SD). Comparisons between two groups were calculated by Student's t test and comparisons among multiple groups were analyzed by ANOVA. The hazards ratio (HR) was calculated with Cox regression. P values less than 0.05 were regarded as statistically significant. The correlation analysis was performed with online tool Sangerbox (http://sangerbox.com/).

DATA AVAILABILITY
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.